The abbreviations shown below are used herein.
AD: transcriptional activation domain
ADH: alcohol dehydrogenase
APC: adenomatous poliposis coli
BD: DNA-binding domain
β-catenin/TCF-4: complex between β-catenin and TCF-4
DCC: deleted in colorectal cancer
dhfr: dihydrofolate reductase
DLG: Drosophila Discs Large
DMSO: dimethylsulfoxide
EGTA: ethylenediaminetetraacetic acid
ELISA: enzyme-linked immunosorbent assay
EST: expressed sequence tag
FAP: familial adenomatous poliposis
FCS: fetal calf serum
FITC: fluorescein isothiocyanate
GST: glutathione S-transferase
GST/ICAT: fusion protein between GST and ICAT
GSK-3β: glycogen synthase kinase-3β
ICAT: inhibitor of β-catenin and TCF
IPTG: isopropylthiogalactoside
KLH: keyhole limpet hemocyanin
Lef: lymphocyte enhancer-binding factor
LTR: long terminal repeat
MBS: m-maleimidobenzoyl-N-hydoroxysuccinimide
MEM: minimum essential medium
PCR: polymerase chain reaction
PEG: polyethylene glycol
RITC: rhodamine isothiocyanate
SDS: sodium dodecyl sulfate
SDS-PAGE: SDS-polyacrylamide gel electrophoresis
TCF: T cell factor
Tris: tris (hydroxymethyl) aminomethane
X-gal: 5-bromo-4-chloro-3-indolyl-β-D-galactoside
The APC gene was isolated as a causative gene of FAP (Kinzler and Vogelstein Cell, 87, 159 (1996)). However, it has been reported that the abnormality of the APC gene is observed not only in FAP but also in 70 to 80% cases of sporadic colon cancer. The onset of colon cancer have been considered to result from successive mutations in many genes including K-ras, p53, DCC and others as well as APC. Mutations are found at the earliest stage in the APC gene as compared with other genes, and thus it has been believed that the abnormality of the APC gene is a primary event for the onset of colon cancer.
In order to clarify the mechanism underlying carcinogenesis associated with the APC gene abnormality, it is necessary to determine the functions of the gene product, APC protein. APC protein, which is about 300 kDa in size, has been reported to bind with β-catenin, glycogen synthase kinase-3β (GSK-3β), as well as DLG in cells (Rubinfeld et al., Science, 262, 1731 (1993); Su et al., Science, 262, 1734 (1993); Rubinfeld et al., Science, 272, 1023 (1996); Matsumine et al., Science, 272, 1020 (1996)). Regarding functions of APC protein, it has been reported that the level of β-catenin is rapidly reduced when wild-type APC protein is expressed in colon cancer cell line SW480 having mutations in the APC gene (Munemitsu et al., Proc. Natl. Acad. Sci. USA, 92, 3046 (1995)). The central region containing a 7-repetitive structure is essential for the function of APC protein and coincides with a region where mutations are found in many colon cancer cases. It has also been reported that the β-catenin level is elevated in these colon cancer cells (Munemitsu et al., Proc. Natl. Acad. Sci. USA, 92, 3046 (1995); Rubinfeld et al., Cancer Res., 57, 4624 (1997)).
β-Catenin is also known as a membrane-skeletal protein for cell adhesion molecule cadherin and also reported to participate in the signal transduction of Wnt protein described below (Cadigan & Nusse, Genes Dev., 11, 3286 (1997)). Wnt gene is a large gene family of which members have a variety of functions in the processes of early embryogenesis and morphogenesis of animals; the family consists of about 20 types of genes in mouse and the genes are conserved among a variety of animals including African clawed frog (Xenopus laevis), fruit fly (Drosophila melanogaster), and nematoda (Caenorhabditis elegans). When Wnt protein binds to a receptor Frizzled, the activity of glycogen synthase kinase-3β (GSK-3β) is inhibited through an intracellular signaling molecule Dishvelled (Dsh). Since the phosphorylation of β-catenin mediated by GSK-3β causes the degradation of β-catenin, the inhibition of GSK-3β activity results in accumulation of β-catenin in cells. β-Catenin binds to a protein belonging to the transcription factor Lef/TCF family to form a complex and thereby activates the protein belonging to the Lef/TCF family as a transcription factor. Thus the accumulation of β-catenin results in formation of the β-catenin/TCF complex, which translocates to the nucleus and thereby stimulates the transcription of target genes. Among proteins belonging to the Lef/TCF family, TCf-4 is specifically expressed in the epithelium of colon, and thus it is believed that β-catenin chiefly forms a complex with TCf-4 in colon cancer (Korinek et al., Science, 275, 1784 (1997)). In addition, it has been reported that there are some colon cancer cells and melanoma cells where the APC gene is wild-type but the β-catenin gene has mutation and is not regulated by GSK-3β (Morin et al., Science, 275, 1787 (1997); Rubinfeld et al., Science, 275, 1790 (1997)). It has been estimated that, in these cells, β-catenin constantly accumulates, which results in transcriptional activation by the β-catenin/TCF complex.
Based on the above-described findings, β-catenin can be greatly involved in the onset of colon cancer. Therefore a substance capable of inhibiting the function of β-catenin through binding thereto can be associated with the onset of colon cancer and thus is predicted to be useful for the treatment, diagnosis, and such thereof. A protein molecule capable of binding to β-catenin, which was recently found, is Axin that negatively regulates the signal transduction of Wnt (Zeng et al., Cell, 90, 181 (1997)). Axin binds to GSK-3β and thereby stimulating the phosphorylation of β-catenin (Ikeda et al., EMBO J., 17, 1371 (1998)). Further it has been reported that Axin also binds to APC and β-catenin to stimulate the degradation of β-catenin and thereby lowering the level of β-catenin in cells (Kishida et al., J. Biol. Chem., 273, 10823 (1998); Rubinfeld et al., Current Biology, 8, 573 (1998); Nakamura et al., Genes to Cells, 3, 395 (1998)). However, there is no known proteins that binds to β-catenin and directly influences the activity of the β-catenin/TCF complex as a transcription factor.